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Shameka J. Shelby, Kecia L. Feathers, Debra A. Thompson; Mertk-Mediated Phospho-tyrosine Signaling in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3351.
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© ARVO (1962-2015); The Authors (2016-present)
Mertk is essential for the phagocytosis of shed photoreceptor outer segment membranes by the retinal pigment epithelium (RPE). The purpose of this study is to identify proteins activated downstream of Mertk by comparing the tyrosine phosphorylated proteins present in the RPE/choroid of congenic and dystrophic Royal College of Surgeons (RCS) rats before and after light onset.
Dissected RPE/choroid was obtained from groups of 3 dystrophic and 3 congenic rats at 30 days of age, at 1.5 h before and 1.5 h after light onset, to generate a total of 4 independent samples. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis, followed by transfer onto PVDF membranes, and probed with a monoclonal phosphotyrosine antibody to detect tyrosine-phosphorylated proteins. Identities of proteins of interest were obtained by MALDI-mass spectrometry (MS) and peptide mass fingerprinting. Expression in the RPE was examined using RT-PCR and western analysis with C57BL/6 mice, and immunohistochemical analysis with Balb/C mice.
At 1.5 h after light-onset, a time corresponding to peak phagocytic uptake by rodent RPE/choroid, more than 25 tyrosine phosphorylated proteins were detected. Of the proteins characterized by MALDI-MS analysis, 5 were intensely phosphorylated after light onset in both congenic and dystrophic animals. These were identified as transthyretin, annexin 4a/actin, tubulin beta 5, selenium-binding protein 1, and rab gdiα. Only rab gdiα was phosphorylated after light onset in congenic animals but not in dystrophic animals. Transcripts encoding all 5 proteins were detected in mouse RPE total RNA. Expression of annexin 4a and rab gdiα were confirmed by western analysis of rat and mouse RPE/choroid protein homogenates, and by immunohistochemical analysis of cryosections.
Following light onset, a number of proteins in the RPE/choroid are tyrosine phosphorylated in the presence or absence of Mertk. Mertk activation appears to be required for tyrosine phosphorylation of rab gdiα that functions as an effector of the Rab family of small GTPases. Thus, Mertk signaling in the RPE may act to regulate proteins that aid in cargo sorting, coordinating vesicle motility, or SNARE-mediated fusion.
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